Modification of intracellular metabolism by expression of a C-terminal variant of phosphoribulokinase from Synechocystis sp. PCC 6803

Abstract Phosphoribulokinase (PRK) is a key enzyme in the Calvin cycle of cyanobacteria required for CO2 fixation and enhancing intracellular PRK activity will contribute to altering the metabolic state. In Synechocystis sp. PCC 6803, PRK activity is inhibited by the small protein CP12 and intramolecular disulfide bonds in its C-terminal loop. This study aimed to increase PRK activity by expressing a mutant PRK that inhibitory Cys residues (positions 229 and 235) in the C-terminal loop were replaced with Ser. The engineered strain showed increased PRK activity under photomixotrophic conditions. Metabolomic analysis revealed that this strain accumulates organic acids downstream of glycolysis and the tricarboxylic acids cycle, highlighting its potential for producing chemicals using these metabolites as precursors. These findings suggest that preventing disulfide bond formation in the PRK C-terminal loop enhances its activity, providing a promising approach for metabolic engineering in cyanobacteria.